Substance transportation between inside and outside of a cell is carried out through transmembrane proteins. It is known that among the transmembrane proteins, the ion channel causes a change in a membrane potential by permeation of ions, so that the ion channel plays an important role in information transmission by the generation of signals, such as a nerve action potential. Therefore, in recent years, there has been an increase in research into the ion channel.
An experimental method called a patch-clamp method is essential for the ion channel research, and the patch-clamp method was developed by Neher and Sakmann in 1976. In the patch-clamp method, first, a tip end of a minute glass tube called a patch electrode is caused to be in close contact with the surface of a cell membrane. A minute membrane region of an opening of the tip end of the minute glass tube is voltage-clamped with the minute membrane region electrically insulated from the other regions, and an ion current flowing through the ion channels contained in the minute membrane region is measured. The development of this method was useful for the identification of a functional element of a channel protein molecule and the elucidation of an operation mechanism and structure of the functional element. Thus, the patch-clamp method has brought significant innovations in physiological research.
Although the patch-clamp method is a highly effective method in the physiological research as described above, there are some cases to which the patch-clamp method cannot be applied. An examples of such case is a case where an access is anatomically difficult, i.e., a case of analyzing the channel on a minute structure, such as a channel on a cell organelle or a presynaptic membrane. In addition, the patch-clamp method is not applicable to a case where an experiment needs to be carried out with a simple configuration in order to advance research into the basic structure of the channel and the relationship between the detailed structure and function of the channel. In this case, the channel molecule needs to be analyzed by a simple system, i.e., a system composed of water, salt, phosphatide, and the channel.
As an effective method used when the patch-clamp method cannot be used, a lipid planar membrane method was developed. The lipid planar membrane method can be broadly classified into a foam spraying method and an attaching method (see NPL 1 for example).
FIG. 18 shows a conventional method for forming the artificial lipid membrane by the foam spraying method. In FIG. 18, a container 10 is divided by a flat plate 11 made from resin, such as Teflon (trademark) or polystyrene, having a hydrophobic surface, a space divided by the flat plate 11 is filled with an electrolytic solution 12, and a lipid solution 14, i.e., a liquid mixture of lipid molecules and an organic solvent is applied by a pipette 15 to a minute hole 13 opening on the flat plate 11. The excess organic solvent contained in the lipid solution 14 applied to the minute hole 13 gradually moves to a peripheral edge of the minute hole 13 to be removed. Thus, the artificial lipid membrane is formed in about 30 minutes to 3 hours.
When forming the artificial lipid membrane, saturated hydrocarbon, such as decane, hexadecane, or hexane, is typically used as the organic solvent. Phosphatide is typically used as the lipid. For example, diphytanoylphosphatidylcholine, or glycerol monooleate, is used.
FIGS. 19(a), 19(b), and 19(c) show another conventional method for forming the artificial lipid membrane by the attaching method. In FIG. 19(a), a container 20 is divided by a flat plate 21 made from resin, such as Teflon (trademark) or polystyrene, having a hydrophobic surface. As a pretreatment, squalene is applied to a minute hole 22 opening on the flat plate 21. An electrolytic solution 23 is added through an inlet 24 to one chamber of the container 20 such that the height of a liquid level of the electrolytic solution 23 does not exceed the height of a lower end of the minute hole 22. Next, a lipid solution, i.e., a liquid mixture of lipid molecules 25 and an organic solvent is dropped from above the container 20 to the electrolytic solution 23, and the solution is left for several minutes. As shown in FIG. 19(a), a lipid monomolecular membrane is formed at a gas-liquid interface of the electrolytic solution 23. The lipid molecule 25 has a hydrophilic portion and a hydrophobic portion, and the hydrophilic portion of the lipid molecule 25 is oriented toward the electrolytic solution 23.
Then, as shown in FIG. 19(b), the electrolytic solution 23 is added through the inlet 24 until the height of the liquid level of the electrolytic solution 23 exceeds the height of an upper end of the minute hole 22.
Next, the same operations as above are carried out in another chamber of the container 20. That is, an electrolytic solution 26 is added through an inlet 27 such that the height of the liquid level of the electrolytic solution 26 does not exceed the height of the lower end of the minute hole 22. Next, the lipid solution is added from above the container 20 to the electrolytic solution 26, and the solution is left for several minutes. The lipid monomolecular membrane is formed at the gas-liquid interface of the electrolytic solution 26. Then, the electrolytic solution 26 is added through the inlet 27 until the height of the liquid level of the electrolytic solution 26 exceeds the height of the upper end of the minute hole 22. By the above operations, the lipid monomolecular membrane formed later is attached to the lipid monomolecular membrane formed in advance at the minute hole 22. As a result, the artificial lipid membrane is formed at the minute hole 22.
However, forming the artificial lipid membrane stably and reproducibly by each of the above-described two methods requires a high degree of skill. As an easier method for forming the artificial lipid membrane, a method for utilizing a MEMS (Micro Electro Mechanical Systems) technology, or a semiconductor microfabrication technology to form the artificial lipid membrane on a small chip was devised (see PTL 1 for example).
FIG. 20 shows a conventional artificial lipid membrane forming apparatus described in PTL 1. In FIG. 20, a first chamber 31 and a second chamber 33 isolated from the first chamber 31 by a dividing wall 32 are provided. The dividing wall 32 has at least one small hole 34 which allows fluidic communication between the first chamber 31 and the second chamber 33. The artificial lipid membrane is formed in the following manner using the artificial lipid membrane forming apparatus shown in FIG. 20. First, the first chamber 31 is filled with a first aqueous solution, and the second chamber 33 is then filled with the lipid solution. The first aqueous solution and the lipid solution are caused to contact each other through the small hole 34. Further, the lipid solution in the second chamber 33 is replaced with a second aqueous solution. With this, an artificial lipid membrane 35 can be formed at the small hole 34.
Another artificial lipid membrane forming apparatus is disclosed in PTL 2. This artificial lipid membrane forming apparatus includes a third introducing port through which the lipid solution is introduced to a micro passage and a first introducing port and a second introducing port through which a first electrolytic solution and a second electrolytic solution, each containing a substance, such as a biologically-relevant substance, are introduced to a micro channel. Then, a molecular membrane is formed at an interface between the first electrolytic solution and the second electrolytic solution.
Further, still another artificial lipid membrane forming apparatus is disclosed in PTL 3. This artificial lipid membrane forming apparatus forms an artificial lipid membrane by covering a minute hole formed on a substrate. In this case, the artificial lipid membrane forming apparatus utilizes a closure phenomenon of the minute hole by a solvent to form the artificial lipid membrane. That is, the membrane is formed in such a state that the lipid solution is supplied onto the substrate on which the minute hole is formed, the substrate swells by the solvent, and the minute hole is closed. After that, the minute hole is opened by the evaporation of the solvent, and the artificial lipid membrane formed is extended. This artificial lipid membrane forming apparatus carries out a minute flow operation and causes the liquid mixture and the electrolytic solution to move at the interface.
Citation List
Patent Literature
PTL 1: Japanese Patent Laid-Open Publication No. 2005-098718 (page 15, FIG. 5)
PTL 2: Japanese Patent Laid-Open Publication No. 2005-185972 (page 73, FIG. 1)
PTL 3: Japanese Patent Laid-Open Publication No. 2005-245331 (page 14, FIG. 2)
Non Patent Literature
NPL 1: “Patch Clamp Experimental Technique” written by Yasunobu Okada, published on Sep. 25, 1996 by Yoshioka Book Store (pages 133-139)